IDH1 R132H Mutation Research Articles

IDH1R132H mutation increases radiotherapy efficacy and a 4-gene radiotherapy-related signature of WHO grade 4 gliomas

The prognosis for the WHO grade 4 IDH-mutant astrocytoma is better than IDH-wildtype glioblastoma (GBM) patients. The purpose of this study is to explore the potential mechanism of how IDH1 mutation can increase the efficacy of radiotherapy and to establish a risk-score model to predict the efficacy of radiotherapy in WHO grade 4 gliomas. First, we conducted experimental study on the effect of IDH1R132H mutation on glioma cells in vitro. Radiosensitivity of glioma cells was detected by γ-H2AX after 5 Gy radiation. Cell proliferation, migration and invasion were determined respectively by CCK-8, EDU, monolayer cell migration scratch assay and Transwell assay. Then we analyzed IDH1 gene status and the survival of WHO grade 4 glioma patients received radiotherapy in our center and verified our results by analyzing CGGA and TCGA database. For the risk-score model, we use CGGA data to find genetic differences between WHO grade 4 IDH-mutant astrocytoma and IDH-wildtype GBM patients, and determined a 4-gene radiotherapy-related signature through survival analysis by R software. Evaluation and verification through different glioma validation sets and different statistical methods. For in vitro experiments, we established glioma cells stably overexpressing IDH1 wild-type and IDH1-mutant proteins. γ-H2AX assay showed that IDH1-mutant glioma cells had higher radiosensitivity than wild-type. CCK-8 and EDU assay showed that proliferation capacity of IDH1-mutant glioma cells declined. Transwell assay and monolayer cell migration scratch assay also showed that IDH1-mutant glioma cells reduced migration and invasion capabilities. Among the 83 WHO grade 4 glioma patients who received radiotherapy in our center, WHO grade 4 IDH-mutant astrocytoma patients had longer OS and PFS versus IDH-wildtype GBM (P = 0.0336, P = 0.0324, respectively). TCGA and CGGA database analysis had the similar results. Through complex analysis of CGGA and TCGA databases, we established a risk-model that can predict the efficacy of radiotherapy for WHO grade 4 glioma patients. The 4-gene radiotherapy-related signature including ADD3, GRHPR, RHBDL1 and SLC9A9. Patients in the high-risk group had worse OS compared to low-risk group (P = 0.0001). High- and low-risk groups of patients receiving radiotherapy have significant survival differences, while patients who did not receive radiotherapy have no survival difference both in CGGA and TCGA databases. WHO grade 4 IDH-mutant astrocytoma is more radiosensitive than IDH-wildtype GBM patients. Our 4-gene radiotherapy-related signature can predict the radiation efficacy of WHO grade 4 glioma patients, and it may provide some reference for clinical treatment options.

CTIM-14. PHASE 1 SAFETY LEAD-IN AND RANDOMIZED OPEN-LABEL PERIOPERATIVE STUDY OF VORASIDENIB COMBINED WITH PEMBROLIZUMAB IN RECURRENT OR PROGRESSIVE ENHANCING IDH1-MUTANT ASTROCYTOMAS: SAFETY LEAD-IN RESULTS

Abstract Isocitrate dehydrogenase 1 mutations (mIDH1) occur in ~80% of grade 2/3 gliomas. Vorasidenib (VOR), an oral, brain-penetrant, dual inhibitor of mIDH1/2 enzymes, is an investigational treatment for mIDH gliomas. A neoadjuvant perioperative study in grade 2/3 mIDH gliomas suggested VOR renders the tumor immune microenvironment amenable to immune checkpoint blockade. This study (NCT05484622) evaluates safety/tolerability of VOR plus pembrolizumab to determine the recommended combination dose (RCD) (safety lead-in phase), and CD3+ T-cell infiltration in tumors following preoperative treatment (perioperative phase). Seven patients with recurrent or progressive enhancing grade 2/3 astrocytoma with an IDH1-R132H mutation were dosed in the safety lead-in phase: median age 39 (range, 34–60) years, 3 (43%) female, 6 (86%) grade 3 at screening. Patients received VOR 40 mg orally once daily (QD) plus pembrolizumab 200 mg intravenously once every 3 weeks (Q3W). As of April 29, 2023, patients received a median of 2 (range, 2–5) cycles with six (86%) ongoing; one (14%) discontinued due to disease progression. No dose-limiting toxicities (DLTs), adverse events (AEs) leading to study drug discontinuation, or AEs of special interest (AESI, elevated liver transaminases) were reported during the DLT evaluation period. Six (86%) patients experienced a treatment-related AE, none of which were grade ≥ 3. One (14%) patient experienced a serious, related AE of dysphasia (leading to VOR and pembrolizumab interruption) that subsequently resolved; both drugs were resumed. After the data cut-off, one patient (14%) experienced a grade 2 AESI. Based on safety review committee evaluation, VOR 40 mg QD + pembrolizumab 200 mg Q3W was confirmed as the RCD for the recently initiated perioperative phase. In this phase, ~60 patients will be randomized 1:1:1 to the RCD, VOR 40 mg QD alone, or no treatment prior to surgery. All patients can receive the RCD post-surgery. Funding: Servier and Merck & Co., Inc.

PATH-27. DROPLET DIGITAL PCR ENHANCES DETECTION OF HOTSPOT MUTATIONS IN CIRCULATING TUMOR DNA FROM CEREBROSPINAL FLUID OF PATIENTS WITH HIGH-GRADE GLIOMA

Abstract Molecular profiling of circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) has the potential to enhance diagnosis, prognosis, and longitudinal monitoring for glioma patients through a minimally invasive approach. We previously detected somatic mutations from gliomas in CSF samples using MSK-IMPACT™, a 505-gene FDA-authorized next-generation DNA sequencing panel, and detected tumor-derived mutations in 50% of samples of patients with high-grade gliomas (HGG). To investigate whether we could enhance sensitivity of CSF ctDNA profiling and biomarker detection, we tested MSK-IMPACT™ pre-capture NGS residual DNA libraries from 43 CSF ctDNA samples by droplet digital PCR (ddPCR) for detection of key recurrent alterations in IDH1, BRAF, TERT, H3F3A, and TP53 using previously validated probes. Residual libraries contained a median of 18.0ng of DNA library (range, 0.0153-18.0ng). Of the 7 samples positive for IDH1 R132H, TERT C228T, or TERT C250T mutations in MSK-IMPACT™, we found that ddPCR accurately detected all mutations (7/7, 100%), confirming sensitivity of ddPCR in CSF ctDNA. We also tested 36 mutation-negative samples by MSK-IMPACT™ for the corresponding undetected mutations, of which 7 were tested for two mutations. Overall, ddPCR detected 41.9% (18/43) of previously undetected mutations. Specifically, we found that ddPCR rescued hotspot mutations such as BRAF V600E (2/3, 66.7%), IDH1 R132G/H (3/8, 37.5%), TERT C228T (11/26, 42.3%), and TERT C250T (2/4, 50.0%). Collectively, our results suggest that a combined-modality approach involving MSK-IMPACT™ and ddPCR enables comprehensive assessment of limited CSF ctDNA samples with high sensitivity and provides a minimally invasive methodology to molecularly profiling glioma.

IMMU-15. INTERPLAY BETWEEN ATRXAND IDH1 MUTATIONS GOVERNS INNATE IMMUNE RESPONSES IN DIFFUSE GLIOMAS

Abstract Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in alpha-thalassemia retardation X-linked (ATRX) are defining molecular alterations in IDH-mutant astrocytomas and have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this interaction, we used both CRISPR and the RCAS/tva system to generate murine models of ATRX-deficient glioma, in the presence and absence of the IDH1 R132H mutation. We found that ATRX-deficient glioma cells were exquisitely sensitive to dsRNA-based innate immune agonism, displaying increases in inflammatory gene expression and profound cytokine release in vitro, while mice bearing ATRX KO tumors displayed increased T-cell infiltration and improved survival. The presence of IDH1 R132H dampened baseline expression of key innate immune genes and cytokines, with genetic and pharmacological inhibition of IDH1 R132H partially rescuing this suppression. Despite the suppression observed at baseline, IDH1 R132H expression did not interfere with the ATRX KO-mediated sensitivity to dsRNA agonism. These results demonstrate that ATRX loss primes cells for recognition of and activation against dsRNA, while IDH1 R132H reversibly masks this priming. Thus, this study suggests that innate immunity is a potential therapeutic vulnerability of astrocytoma. Further studies are in progress to validate the influence of ATRX loss on immune signaling and tumor-immune coevolution within human tumors.

TMIC-07. MUTANT IDH-MEDIATED SUPPRESSION OF THE TH17 LINEAGE IN GLIOMA; BIOLOGIC IMPACT AND THERAPEUTIC POTENTIAL

Abstract Great excitement has surrounded the identification of tumor-specific hotspot mutations in IDH1 as they are the first therapeutic targets of low grade gliomas that have been identified in the last several decades. With a low number of viable therapeutic targets, mutations in IDH1 represent new hope in treating these tumors that are invariably recurrent and confer a dismal prognosis in their aggressive forms. Additionally, mutations in IDH1 are tumor-specific suggesting their effective targeting can reduce the collateral damage to the normal brain imparted by standard therapies. Furthermore, mutations in IDH1 lead to the overproduction of D-2-hydroxyglutarate (D-2HG), which mediates many of mutant IDH1’s effects. To gain an understanding of mutant IDH-mediated tumorigenesis, we generated genetically faithful mouse models that harbor the pathognomonic signature including the IDH1-R132H mutation and p53 deletion. This model generates tumors that overproduce D-2HG, are histologically diffuse, and confer a prolonged survival compared to their wildtype counterparts. Additionally, these models display the proneural subtype and are enriched for an oligodendrocyte precursor cell gene expression signature, both of which are typified by IDH mutations in human glioma. Exploring the impact of the IDH mutation on the tumor microenvironment, we identified several diminished immune cell subsets among the mutant IDH murine tumors, including NK cells, monocytes, and M2 macrophages. Further analysis showed a reduced Th17 gene expression profile that was corroborated through in vitro studies. The biological impact of Th17 cells appears to be cancer-context dependent with only a few studies being performed in gliomas. With the potential for Th17-mediated anti-tumor activity, as observed in cases of melanoma, the prospect for elucidating a new therapeutic avenue is exciting. As such, the biological significance of D-2HG-mediated Th17 suppression, the underlying mechanism, as well as the therapeutic potential of Th17 restoration are actively being investigated.

Rapid extraction-free detection of the R132H isocitrate dehydrogenase mutation in glioma using colorimetric peptide nucleic acid-loop mediated isothermal amplification (CPNA-LAMP).

The R132H isocitrate dehydrogenase one (IDH1) mutation is a prognostic biomarker present in a subset of gliomas and is associated with heightened survival when paired with aggressive surgical resection. In this study, we establish proof-of-principle for rapid colorimetric detection of the IDH1-R132H mutation in tumor samples in under 1 hour without the need for a nucleic acid extraction. Colorimetric peptide nucleic acid loop-mediated isothermal amplification (CPNA-LAMP) utilizes 4 conventional LAMP primers, a blocking PNA probe complementary to the wild-type sequence, and a self-annealing loop primer complementary to the single nucleotide variant to only amplify the DNA sequence containing the mutation. This assay was evaluated using IDH1-WT or IDH1-R132H mutant synthetic DNA, wild-type or IDH1-R132H mutant U87MG cell lysates, and tumor lysates from archived patient samples in which the IDH1 status was previously determined using immunohistochemistry (IHC). Reactions were performed using a hot water bath and visually interpreted as positive by a pink-to-yellow color change. Results were subsequently verified using agarose gel electrophoresis. CPNA-LAMP successfully detected the R132H single nucleotide variant, and results from tumor lysates yielded 100% concordance with IHC results, including instances when the single nucleotide variant was limited to a portion of the tumor. Importantly, when testing the tumor lysates, there were no false positive or false negative results.

P14.12.A PLASMA CFDNA LIQUID BIOPSY IN THE FOLLOW-UP OF HIGH-GRADE GLIOMAS

Abstract BACKGROUND As highlighted by the 2021 WHO classification of brain tumors, molecular characterization of gliomas is now critical to complement histopathological evaluation. However, intratumor heterogeneity may lead to surgical specimens not being fully representative of the whole tumor. Moreover, given the invasiveness of surgery, tissue analyses are not repeatable over time, thus limiting the possibility to monitor the response to therapies. In this context, we investigated the potential value of plasmatic cell-free DNA (PcfDNA) as a non-invasive ‘liquid biopsy’ marker of disease evolution. MATERIAL AND METHODS Patients’ blood was collected at 4 time points (TP), in parallel to clinical follow-up. A reproducible pipeline was optimized for blood withdrawal, centrifugation, plasma storage, cfDNA extraction from plasma by QIAamp MinElute ccfDNA Midi Kit (Quiagen) and cfDNA fluorometric quantification by Qubit (Thermo Fisher Scientific). MRI volumes were segmented in T1+contrast and T2-FLAIR sequences. RESULTS Fifty-four high-grade glioma patients and 14 healthy controls were enrolled. Blood samples were collected from all 54 patients at radiological diagnosis before surgery (TP0), from 33 patients after surgery (TP1), from 21 patients after radio-chemotherapy(TP2), from 10 patients at the first radiological progression (TP3). At T0, a significantly higher concentration of PcfDNA was detected in patients than in controls. IDH-wild-type gliomas showed a trend toward higher PcfDNA when compared with IDH-mutant gliomas. Digital-droplet PCR, performed at T0on PcfDNA of 4 patients with IDH-mutations in tumor tissues, identified the same IDH1-R132H mutation in all cases. When PcfDNA at TP0 was compared to that at TP1 of the same patient (n=33), there was a significant reduction of plasmatic cfDNA after surgery, mirroring the extensive tumor-resection shown by MRI. Finally, PcfDNA was evaluated in 10 patients until their radiological progression. Interestingly, PcfDNA levels not only followed the same trend of the volumes of T2-FLAIR hyperintensities and T1 contrast-enhancements, but were also mirrored by the evolution of KPS and NANO scores. CONCLUSION Preliminary results suggest that PcfDNA content may be an informative tool to complement MRI during the follow-up of high-grade gliomas.

P12.05.B THE ROLE OF ONCO-METABOLITE IN THE IDH MUTANT GLIOBLASTOMA MICROENVIRONMENT

Abstract BACKGROUND Recently, we found that the reactive astrocytes in the IDH wild type glioblastoma contribute to anti-tumor immunity and support pro-oncogenic signaling. Here, we characterized the transcriptomic signature of IDH1/2-mutant glioma associated astrocytes and determined a unique inflammatory transformation, profoundly different to astrocytes in IDH wildtype glioma patients due to an onco-metabolite R-2-hydroxyglutarate using Next generation sequencing and human ex-vivo slice model. MATERIAL AND METHODS We purified and transcriptionally profiled astrocytes from 9 patients with confirmed IDH1-R132H mutation, by means of RNA-sequencing and the data were analyzed using the established pipelines. We also used spatial transcriptomics to evidently show the spatial distribution of astrocytes in IDH-mutated/wildtype glioma samples. We validated our findings using human organotypic slice model inoculated with IDH-mutant cell line or treated with oncometabolite 2-hydroxy glutarate. Additionally, LC-MS was further used to give us a chart of neurotransmitters due to altered microenvironment. RESULTS Our results from RNA sequencing showed a transcriptional transformation of reactive astrocytes within the microenvironment of IDH-mutated tumors compared to wildtype glioma by means of RNA-sequencing of purified astrocytes. And, using our established human neocortical GBM model inoculated with IDH mutant tumor and R-2HG treatment, we showed that we were able to activate inflammatory transcriptional programs in astrocytes, mediated by the presence of microglia. Further, by spatially mapping the transcriptomic profiles of purified microglia, we were able to confirm that microglia also demonstrate inflammatory activation in IDH mutated glioma. This inflammatory astrocyte transformation is associated with a loss of neurotransmitter homeostasis (disrupted levels of glutamate) in the treated sections, as has been previously reported in IDH mutated tumors. Additionally, R-H2G increased neuronal spiking rate in, pointing to potential excitotoxicity. CONCLUSION Our work provides a crucial contribution towards understanding the role of R-2HG in the IDH mutant glioma microenvironment and sheds light on the significant microenvironmental differences to IDH wild-type glioma.

Diagnostic Utility of Immunohistochemical Detection of MEOX2, SOX11, INSM1 and EGFR in Gliomas.

Histological identification of dispersed glioma cells in small biopsies can be challenging, especially in tumours lacking the IDH1 R132H mutation or alterations in TP53. We postulated that immunohistochemical detection of proteins expressed preferentially in gliomas (EGFR, MEOX2, CD34) or during embryonal development (SOX11, INSM1) can be used to distinguish reactive gliosis from glioma. Tissue microarrays of 46 reactive glioses, 81 glioblastomas, 34 IDH1-mutant diffuse gliomas, and 23 gliomas of other types were analysed. Glial neoplasms were significantly more often (p < 0.001, χ2) positive for EGFR (34.1% vs. 0%), MEOX2 (49.3% vs. 2.3%), SOX11 (70.5% vs. 20.4%), and INSM1 (65.4% vs. 2.3%). In 94.3% (66/70) of the glioblastomas, the expression of at least two markers was observed, while no reactive gliosis showed coexpression of any of the proteins. Compared to IDH1-mutant tumours, glioblastomas showed significantly higher expression of EGFR, MEOX2, and CD34 and significantly lower positivity for SOX11. Non-diffuse gliomas were only rarely positive for any of the five markers tested. Our results indicate that immunohistochemical detection of EGFR, MEOX2, SOX11, and INSM1 can be useful for detection of glioblastoma cells in limited histological samples, especially when used in combination.

Correlating MRI features with additional genetic markers and patient survival in histological grade 2-3 IDH-mutant astrocytomas

PurposeThe increasing importance of molecular markers for classification and prognostication of diffuse gliomas has prompted the use of imaging features to predict genotype (“radiogenomics”). CDKN2A/B homozygous deletion has only recently been added to the diagnostic paradigm for IDH[isocitrate dehydrogenase]-mutant astrocytomas; thus, associated radiogenomic literature is sparse. There is also little data on whether different IDH mutations are associated with different imaging appearances. Furthermore, given that molecular status is now generally obtained routinely, the additional prognostic value of radiogenomic features is less clear. This study correlated MRI features with CDKN2A/B status, IDH mutation type and survival in histological grade 2-3 IDH-mutant brain astrocytomas.MethodsFifty-eight grade 2–3 IDH-mutant astrocytomas were identified, 50 with CDKN2A/B results. IDH mutations were stratified into IDH1-R132H and non-canonical mutations. Background and survival data were obtained. Two neuroradiologists independently assessed the following MRI features: T2-FLAIR mismatch (<25%, 25–50%, >50%), well-defined tumour margins, contrast-enhancement (absent, wispy, solid) and central necrosis.Results8/50 tumours with CDKN2A/B results demonstrated homozygous deletion; slightly shorter survival was not significant (p=0.571). IDH1-R132H mutations were present in 50/58 (86%). No MRI features correlated with CDKN2A/B status or IDH mutation type. T2-FLAIR mismatch did not predict survival (p=0.977), but well-defined margins predicted longer survival (HR 0.36, p=0.008), while solid enhancement predicted shorter survival (HR 3.86, p=0.004). Both correlations remained significant on multivariate analysis.ConclusionMRI features did not predict CDKN2A/B homozygous deletion, but provided additional positive and negative prognostic information which correlated more strongly with prognosis than CDKN2A/B status in our cohort.

Ivosidenib off-label use for IDH mutant gliomas: The MD Anderson Cancer Center real life experience.

e14022 Background: Ivosidenib, a small-molecule IDH inhibitor is FDA approved for relapsed or refractory acute myeloid leukemia and cholangiocarcinoma with IDH1 mutation. Early phase clinical trials have shown promising findings for IDH mutant gliomas and results of larger studies of IDH inhibitors in gliomas are still pending. Meanwhile, neuro-oncologists continue to encounter patients with IDH mutant gliomas with limited treatment options. Here, we describe our experience with off-label use of ivosidenib in IDH mutant gliomas. Methods: This is a retrospective case series study in a single cancer care institute. Data points collected include but not limited to patient demographics, type of IDH mutation, treatment history prior to starting ivosidenib, duration of treatment, and progression-free survival. Descriptive statistics were used to summarize patient’s characteristics. The distribution of median progression-free survival (mPFS) was estimated by the Kaplan-Meier method. Results: From August 2018 to December 2022, we identified 13 patients with IDH-mutant glioma prescribed ivosidenib 500mg daily off-label. Four patients did not receive treatment: two were too sick soon after ivosidenib prescribed, and two were not able to get financial coverage. For the nine patients that were treated, median age was 37 (range 25-44). 8 were males and 1 was female. Five have IDH1 R132H mutations, 1p19q intact and four have IDH mutation and 1p19q co-deletion. The highest grade for the tumor is grade 3. Four have non-enhancing disease only. The median lines of therapy prior to starting ivosidenib was 2 (range 0-5). Three patients did not have radiation by the time they started ivosidenib, including two who received this drug as first-line treatment initiated by providers outside of MD Anderson. All patients are still alive as of January 11th, 2023. Disease control rate (all stable disease) was 78%. After a median follow-up of 17.8 (range 15-29) months, mPFS was 3.81 (95% CI: 1.25, 12.78) months. Patients with non-enhancing disease had better PFS than those with enhancing disease, but this was not statistically significant (median PFS: 8.28 vs 3.81 months, p-value = 0.42). Two patients had adverse events: one with grade 1 lethargy, and another patient had a grade 3 surgical wound infection unrelated to the drug requiring a pause in treatment. Conclusions: Ivosidenib was successfully approved by third party payers for treatment of nine patients with IDH mutant gliomas. Ivosidenib was well-tolerated with a modest PFS. Patients with non-enhancing disease may trend toward better PFS than those with enhancing disease. The updated survival data and potential correlation with clinical, molecular, and radiographic data will be presented at the conference.

Xenonucleic acid technology in high-sensitivity IDH1/2 mutation detection for glioma molecular profiling.

e14052 Background: Glioma is a life-threatening primary brain tumor that poses significant challenges for diagnosis and treatment. Certain genetic features, such as the IDH1/2 mutation status (codon 132 of IDH1 and codon 172 of IDH2), can help distinguish between different types of gliomas. IDH1/2 wild-type and mutant gliomas have different prognoses and treatment options, with patients with IDH1/2 mutations typically having a better prognosis and a better response to chemotherapy. However, current available molecular tests for IDH1/2 mutation detection have limited sensitivity, and their application to circulating cell-free DNA has not been well established. Methods: To address these challenges, we have developed a new technology that uses xenonucleic acids (XNA) to achieve high sensitivity and specificity for detecting IDH1/2 mutations. We designed and synthesized a specific XNA probe to block PCR amplification from the IDH1 wild-type allele so that the IDH1 mutant allele can be enriched by Taqman real-time PCR. Results: Our tests showed that the XNA-mediated Taqman Real-time PCR system could efficiently detect IDH1-R132H mutations at a frequency as low as 0.1% (e.g., 7 IDH1-R132H copies out of 7,425 IDH1 copies) in both synthetic DNA fragments and genomic DNA from glioma stem cell lines. Clinical verification and validation studies are ongoing using tissue biopsies and paired plasma circulating cell-free DNA (cfDNA). The results of these studies will further demonstrate the reliability and accuracy of our XNA-based detection technology and its potential impact on patient outcomes. Conclusions: Our innovative technology holds the potential to significantly enhance the diagnosis of gliomas and other cancers. It offers a cost-effective, readily available, and precise diagnostic solution that can be performed by standard pathology personnel using equipment already available in hospital pathology laboratories. We will showcase the innovative capabilities of our XNA-based detection technology and its potential to improve the diagnosis and management of gliomas.

Structure-based discovery of IHMT-IDH1-053 as a potent irreversible IDH1 mutant selective inhibitor

Through a structure-based irreversible drug design approach, we have discovered a highly potent IDH1-mutant inhibitor compound 16 (IHMT-IDH1-053) (IC50 = 4.7 nM), which displays high selectivity against IDH1 mutants over IDH1 wt and IDH2 wt/mutants. The crystal structure demonstrates that 16 binds to the IDH1 R132H protein in the allosteric pocket adjacent to the NAPDH binding pocket through a covalent bond with residue Cys269. 16 inhibits 2-hydroxyglutarate (2-HG) production in IDH1 R132H mutant transfected 293T cells (IC50 = 28 nM). In addition, it inhibits the proliferation of HT1080 cell line and primary AML cells which both bear IDH1 R132 mutants. In vivo, 16 inhibits 2-HG level in a HT1080 xenograft mouse model. Our study suggested that 16 would be a new pharmacological tool to study IDH1 mutant-related pathology and the covalent binding mode provided a novel approach for designing irreversible IDH1 inhibitors.

Glioma immunotherapy enhancement and CD8-specific sialic acid cleavage by isocitrate dehydrogenase (IDH)-1

The promise of adaptive cancer immunotherapy in treating highly malignant tumors such as glioblastoma multiforme (GBM) can only be realized through expanding its benefits to more patients. Alleviating various modes of immune suppression has so far failed to achieve such expansion, but exploiting endogenous immune enhancers among mutated cancer genes could represent a more direct approach to immunotherapy improvement. We found that Isocitrate Dehydrogenase-1 (IDH1), which is commonly mutated in gliomas, enhances glioma vaccine efficacy in mice and discerns long from short survivors after vaccine therapy in GBM patients. Extracellular IDH1 directly enhanced T cell responses to multiple tumor antigens, and prolonged experimental glioma cell lysis. Moreover, IDH1 specifically bound to and exhibited sialidase activity against CD8. By contrast, mutant IDH1R132H lacked sialidase activity, delayed killing in glioma cells, and decreased host survival after immunotherapy. Overall, our findings identify IDH1 as an immunotherapeutic enhancer that mediates the known T cell-enhancing reaction of CD8 desialylation. This uncovers a new axis for immunotherapeutic improvement in GBM and other cancers, reveals novel physiological and molecular functions of IDH1, and hints at an unexpectedly direct link between lytic T cell function and metabolic activity in target cells.

Abstract 1261: Mutant IDH-mediated suppression of the Th17 lineage in glioma: Biologic impact and therapeutic potential

Abstract Great excitement has been generated around the identification of tumor-specific hotspot mutations in isocitrate dehydrogenase I (IDH1) as they are the first potential therapeutic targets of low grade gliomas that have been identified in the last several decades. With a low number of viable therapeutic targets, mutations in IDH1 represent new hope in treating these devastating tumors that are invariably recurrent and confer a dismal prognosis in their most aggressive forms. In addition to their high frequency of mutation in gliomas, mutations in IDH1 are tumor-specific suggesting their effective targeting can reduce the collateral damage to the normal brain imparted by standard therapies. Furthermore, mutations in IDH1 lead to the overproduction of the oncometabolite D-2-hydroxyglutarate (D-2HG), which mediates many of mutant IDH1’s effects including blocks to normal differentiation, aberrant histone and DNA methylation profiles, perturbed metabolism, induction of an immunosuppressive microenvironment, among others. To gain an understanding of mutant IDH-mediated tumorigenesis, we generated genetically faithful mouse models of glioma that harbor the pathognomonic signature including the IDH1-R132H mutation and p53 deletion. This orthotopic intracranial injection model generates tumors that overproduce D-2HG, are histologically diffuse, and confer a prolonged survival compared to their wildtype counterparts. Additionally, these models display the proneural glioma subtype and are enriched for an oligodendrocyte precursor cell gene expression signature, both of which are typified by IDH mutations in human glioma. Exploring the impact of the IDH mutation on the tumor microenvironment, we identified several diminished immune cell subsets among the mutant IDH murine tumors, including plasma cells, NK cells, monocytes, and M2 macrophages, while a subtle increase among dendritic cells was observed as indicated through immune deconvolution of RNAseq data of murine brain tumors. Further analysis showed a reduced Th17 gene expression profile that was corroborated through in vitro studies showing a D-2HG-mediated suppression of IL17 producing Th17 cells and a perturbed metabolic activity skewing towards oxidative phosphorylation. Th17 cells have only relatively recently been identified and their biological impact appears to be cancer-context dependent with only a few studies being performed in gliomas. With the potential for Th17-mediated anti-tumor activity, as observed in cases of melanoma, the prospect for understanding this understudied lineage and exposing a new therapeutic avenue is exciting. As such, the biological significance of D-2HG-mediated Th17 suppression, the underlying mechanism, as well as the therapeutic potential and anti-tumor activity of Th17 restoration or IL17 overproduction are actively being investigated. Citation Format: Nathan M. Reynolds, Ashleigh J. Soedel, Ximena Bustamante-Marin, Emily Z. Miller, Paula K. Greer, David M. Ashley, Yiping He, Matthew S. Waitkus, Christopher J. Pirozzi. Mutant IDH-mediated suppression of the Th17 lineage in glioma: Biologic impact and therapeutic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1261.

Detection of IDH1 Mutation in cfDNA and Tissue of Adult Diffuse Glioma with Allele-Specific qPCR.

The World Health Organization (WHO) classification of central nervous system (CNS) tumors necessitates testing of isocitrate dehydrogenase (IDH) 1/2 gene mutation in patients with adult-type diffuse glioma (ADG) for better disease management. In clinical practice, the testing of IDH1 is primarily achieved using immunohistochemistry (IHC) specific to IDH1-R132, which carries a sensitivity of 80% and specificity of 100%. However, in some cases, non-specific background staining or regional heterogeneity in the protein expression of IDH1 may necessitate confirmatory genetic analysis. Robust and reliable assays are needed for IDH1/2 mutation testing. The aim of the current study was to detect IDH1 mutation in cfDNA and tissue of adult-type diffuse glioma with allele-specific qPCR. In the current study, IDH1-R132H mutation was analyzed in tumor tissue with paired cell-free DNA (cfDNA) in patients with ADG (n = 45) using IHC and competitive allele-specific Taqman PCR (CAST-PCR). Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue and matched serum for cfDNA using commercially available kits. CAST-PCR with IHC for the detection of IDH1-R132H mutation was also compared. The IDH1-R132H mutation was detected in 46.67% (21/45) cases and 57.78% (26/45) cases using IHC and allele-specific CAST-PCR. In cfDNA of matched IDH1-mutant FFPE tissue DNA, IDH1-R132H mutation was detected in 11.54% (3/26) using CAST-PCR. The concordance rate for IDH1-R132Hmutation between IHC and CAST-PCR was 80.77% (21/26). The CAST-PCR assay is more precise and sensitive for IDH1-R132Hdetection than traditional IHC, and IDH1-R132H mutation detection using cfDNA may add to the current methods of glioma genomic characterization.

Nanomechanical Signatures in Glioma Cells Depend on CD44 Distribution in IDH1 Wild-Type but Not in IDH1R132H Mutant Early-Passage Cultures

Atomic force microscopy (AFM) recently burst into biomedicine, providing morphological and functional characteristics of cancer cells and their microenvironment responsible for tumor invasion and progression, although the novelty of this assay needs to coordinate the malignant profiles of patients' specimens to diagnostically valuable criteria. Applying high-resolution semi-contact AFM mapping on an extended number of cells, we analyzed the nanomechanical properties of glioma early-passage cell cultures with a different IDH1 R132H mutation status. Each cell culture was additionally clustered on CD44+/- cells to find possible nanomechanical signatures that differentiate cell phenotypes varying in proliferative activity and the characteristic surface marker. IDH1 R132H mutant cells compared to IDH1 wild-type ones (IDH1wt) characterized by two-fold increased stiffness and 1.5-fold elasticity modulus. CD44+/IDH1wt cells were two-fold more rigid and much stiffer than CD44-/IDH1wt ones. In contrast to IDH1 wild-type cells, CD44+/IDH1 R132H and CD44-/IDH1 R132H did not exhibit nanomechanical signatures providing statistically valuable differentiation of these subpopulations. The median stiffness depends on glioma cell types and decreases according to the following manner: IDH1 R132H mt (4.7 mN/m), CD44+/IDH1wt (3.7 mN/m), CD44-/IDH1wt (2.5 mN/m). This indicates that the quantitative nanomechanical mapping would be a promising assay for the quick cell population analysis suitable for detailed diagnostics and personalized treatment of glioma forms.

Characterization of an IDH1 R132H Rabbit Monoclonal Antibody, MRQ-67, and Its Applications in the Identification of Diffuse Gliomas.

The current diagnosis of diffuse glioma involves isocitrate dehydrogenase (IDH) mutation testing. Most IDH mutant gliomas carry a G-to-A mutation at IDH1 position 395, resulting in the R132H mutant. R132H immunohistochemistry (IHC), therefore, is used to screen for the IDH1 mutation. In this study, the performance of MRQ-67, a recently generated IDH1 R132H antibody, was characterized in comparison with H09, a frequently used clone. Selective binding was demonstrated by an enzyme-linked immunosorbent assay for MRQ-67 to the R132H mutant, with an affinity higher than that for H09. By Western and dot immunoassays, MRQ-67 was found to bind specifically to the IDH1 R1322H, with a higher capacity than H09. IHC testing with MRQ-67 demonstrated a positive signal in most diffuse astrocytomas (16/22), oligodendrogliomas (9/15), and secondary glioblastomas tested (3/3), but not in primary glioblastomas (0/24). While both clones demonstrated a positive signal with similar patterns and equivalent intensities, H09 exhibited a background stain more frequently. DNA sequencing on 18 samples showed the R132H mutation in all IHC positive cases (5/5), but not in negative cases (0/13). These results demonstrate that MRQ-67 is a high-affinity antibody suitable for specific detection of the IDH1 R132H mutant by IHC and with less background as compared with H09.

The prognostic impact of subclonal IDH1 mutation in grade 2-4 astrocytomas.

Isocitrate dehydrogenase (IDH) mutations are thought to represent an early oncogenic event in glioma evolution, found with high penetrance across tumor cells; however, in rare cases, IDH mutation may exist only in a small subset of the total tumor cells (subclonal IDH mutation). We present 2 institutional cases with subclonal IDH1 R132H mutation. In addition, 2 large publicly available cohorts of IDH-mutant astrocytomas were mined for cases harboring subclonal IDH mutations (defined as tumor cell fraction with IDH mutation ≤0.67) and the clinical and molecular features of these subclonal cases were compared to clonal IDH-mutant astrocytomas. Immunohistochemistry (IHC) performed on 2 institutional World Health Organization grade 4 IDH-mutant astrocytomas revealed only a minority of tumor cells in each case with IDH1 R132H mutant protein, and next-generation sequencing (NGS) revealed remarkably low IDH1 variant allele frequencies compared to other pathogenic mutations, including TP53 and/or ATRX. DNA methylation classified the first tumor as high-grade IDH-mutant astrocytoma with high confidence (0.98 scores). In the publicly available datasets, subclonal IDH mutation was present in 3.9% of IDH-mutant astrocytomas (18/466 tumors). Compared to clonal IDH-mutant astrocytomas (n = 156), subclonal cases demonstrated worse overall survival in grades 3 (P = .0106) and 4 (P = .0184). While rare, subclonal IDH1 mutations are present in a subset of IDH-mutant astrocytomas of all grades, which may lead to a mismatch between IHC results and genetic/epigenetic classification. These findings suggest a possible prognostic role of IDH mutation subclonality, and highlight the potential clinical utility of quantitative IDH1 mutation evaluation by IHC and NGS.

MPC-10 RECURRENT ASTROCYTOMA WITH SOMATIC IDH MOSAICISM: A CASE REPORT AND WARRANT FOR MOLECULAR DIAGNOSTICS

Abstract Introduction Acquisition of somatic mutations is essential for the development of malignant tumors such as diffuse gliomas. Recent single-cell analyses have pointed out the importance of somatic mosaicism in the pathogenesis of cancer. Here we report a case of IDH (isocitrate dehydrogenase)-mutant astrocytoma that has relapsed after intensive chemoradiation with somatic mosaicism for the IDH1 R132H mutation. Clinical and Pathological Findings A 36-year-old male presented with epileptic symptoms and a mass lesion in the right frontal lobe. A needle biopsy demonstrated IDH-mutant astrocytoma (grade 2). At the age of 41, relapsed tumor was completely resected, and the tumor cells were diffusely positive for IDH1 R132H with grade 4 morphology. Chemoradiotherapy (PAV + IMRT 60Gy/30fr) was added, but contrast-enhanced lesions reappeared 5 months after the initial surgery. The re-excised specimen was grade 4 astrocytoma, and the number of IDH1 R132H-positive tumor cells was very small compared to the total number of tumor cells, suggesting somatic mosaicism for IDH mutations. Conclusion It is imperative to clarify the induction mechanism and significance of somatic cell mosaicism in cancer, and molecular genetic assessment with sequencing and digital PCR would be necessary in addition to immunostaining for accurate molecular diagnoses of the brain tumors.